1. Field of the Invention
The present invention is directed to a method for cycle sequencing of DNA with marker-substituted dideoxynucleotides using extremely thermophilic DNA polymerases as well as a novel use of extremely thermophilic DNA polymerases.
2. Description of the Prior Art
Sequence analysis of DNA has become an irretrievable tool both for basic research and for laboratory diagnostics and is used routinely. Sequencing protocols are almost exclusively based on the chain termination method of Sanger using radiolabeled or fluorescent markers for the labeling of DNA chains. Various companies are offering a plurality of different sequencing protocols. In particular, the protocol of Applied Biosystems company (ABI) for non-radioactive sequencing using dye-labeled dideoxy terminators (dye-dideoxy terminators) has been widely used.
If sequencing is performed using dye-dideoxy terminators, the DNA polymerases copy the DNA template to be sequenced in a primer-dependent manner. Chain termination is accomplished by incorporation of ddNTPs each labeled by a different fluorescent dye. This method enables the separation of the DNA sequence in a single lane whereas the classical DNA sequencing method of Sanger using unlabeled ddNTPs and performing the labeling of the chains by radioactive .alpha.-dATP or fluorescent primers requires a four-lane system and, in the case of one-lane analysis, an elaborate 4-primer method.
If the sequencing reaction is effected in a single cycle at 37.degree. C. generally thermolabile polymerases are used such as the T7 DNA polymerase. For many applications this protocol provides excellent results; however, it has the disadvantage that it requires high amounts of template DNA (about 5 .mu.g of plasmid DNA per reaction). This disadvantage is particularly recognized in the direct sequencing of PCR products. Insurmountable difficulties are frequently encountered if GC-rich templates are sequenced. In contrast, less than 1/5 of the template DNA is necessary in cycle sequencing using DNA polymerases obtained from extremely thermophilic organisms. This method comprises various cycles of denaturation, annealing of the sequencing primer and chain elongation with subsequent ddNTP incorporation. According to the prior art this latter step has been carried out at 60.degree. C. which in the case of the sequencing of GC-rich DNA sequences and of DNA having secondary structures leads to superior results over the protocols using only a single cycle at 37.degree. C. However, the temperature of 60.degree. C. used in the protocols according to the prior art is far below the optimum synthesis temperature of extremely thermostable DNA polymerases.
Unlike the protocol mentioned above, dye primer protocols are able to use an optimum chain elongation temperature of 72.degree. C. However, these protocols require a cost-intensive extra-synthesis of fluorescence-labeled primers: of 1 primer in the case of the 4-lane method, and even of 4 primers if the 1-lane analysis is performed. While dye terminator sequencing is carried out in only one sample, in both of the cases the preparation of 4 parallel reactions is necessary each containing one of the ddNTPs leading to an additional increase in time and costs.
Protocols for dye primer sequencing are described by various companies for different extremely thermostable DNA polymerases. Examples for such protocols are those of ABI/Perkin Elmer, using AmpliTaq.RTM. and AmpliTaqFS.RTM., Amersham using Thermosequenase.RTM. or of Epicentre using Sequitherm.RTM. DNA polymerase.
If the dye primer sequencing is used as a 1-lane technique it is necessary to provide four different primers with different dye labels. However, this accordingly leads to an increase in the time and costs required for sequencing.
Up to now successful cycle sequencing using dye terminators is known from the prior art only at a chain elongation temperature of 60.degree. C., and this has been applied to the AmpliTaq.RTM., AmpliTaqFS.RTM., Thermosequenase.RTM., Sequitherm.RTM., and Tfl DNA polymerase enzymes. To date no protocols have been reported for temperatures in excess of 60.degree. C. for cycle sequencing using dye terminators.